The long-term objective of this proposal is to elucidate the molecular mechanisms that regulate Sertoli cell proliferation and differentiation thereby determining the factors that limit spermatogenic capacity and male fertility. Sertoli cells are essential for the maturation and expansion of spermatozoa but the somatic cell can support only a finite number of germ cells. The size of the Sertoli cell population sets the upper limit for male fertility and is determined by the proliferative capacity of Sertoli cells prior to their terminal differentiation during puberty. The overall hypothesis to be tested is that the relative activities of Id and Ebox proteins expressed by Sertoli cells during development determine whether these somatic cells proliferate or differentiate. Aim 1 is to determine whether Id proteins regulate Sertoli cell proliferation and differentiation. Aim 2 is to determine whether up-regulation of E-box binding activity is required for Sertoli cell differentiation. Aim 3 is to identify the mechanisms employed by Id proteins and E-box transcription factors to regulate Sertoli cell development. Over expression and knockdown of Id proteins using adenoviral vectors and RNA interference (RNAi) in rat and non-human primate (rhesus monkey) Sertoli cell models will be used to determine whether Id proteins directly stimulate Sertoli cell proliferation and inhibit differentiation. E-box mRNA expression, DNA binding activity and promoter stimulating activity will be assessed using quantitative RT-PCR, DNA-protein binding assays and transient transfection assays, respectively. To determine whether Id and E-box proteins regulate genes governing Sertoli cell development, the expression of target genes will be assessed by quantitative RT-PCR after over expression and RNAi knockdown of Id and E-box proteins. Collectively, the results of these studies in both rodent and primate systems will establish the molecular mechanisms that regulate Sertoli cell proliferation and differentiation. Understanding this aspect of post-natal testicular development in both rodent and primate models will provide important insights into the pathophysiology of male infertility.